Practical considerations for the analysis of time-resolved x-ray data

Abstract

The field of time-resolved macromolecular crystallography has been expanding rapidly after free electron lasers for hard x rays (XFELs) became available. Techniques to collect and process data from XFELs spread to synchrotron light sources. Although time-scales and data collection modalities can differ substantially between these types of light sources, the analysis of the resulting x-ray data proceeds essentially along the same pathway. At the base of a successful time-resolved experiment is a difference electron density (DED) map that contains chemically meaningful signal. If such a difference map cannot be obtained, the experiment has failed. Here, a practical approach is presented to calculate DED maps and use them to determine structural models.

Structural Dynamics of Metalloproteins and Redox Enzymology with Mix-and-Inject Time-Resolved Serial Femtosecond Crystallography

“…105,169 In addition, the shot-to-shot heterogeneity, nonisomorphic unit cell parameters, and crystal orientation problems complicate further analysis of the tr-SFX data, leading to difficulties in detecting difference structure factor signals Δ|F| τ . 106 Principal component analysis (PCA) methods such as single value decom-position (SVD) were used previously 170 to analyze timeresolved macromolecular X-ray data using photoactive yellow protein crystals and were successfully applied to pump−probe tr-SFX data. 37,168 SVD allows for unbiased differentiation between signals and noise, thereby enhancing the detection of small difference structure factor signals.…”

“…Recent results on structural enzymology using the MISC tr -SFX technique provide a comprehensive and detailed understanding of the processing and interpretation of MISC data, thus enabling scientists to extract essential structural information on the different transient-kinetic states during the catalytic cycles of these enzymes. ,− MISC tr -SFX enables direct estimation of enzyme kinetics, as previously demonstrated in BlaC β-lactamase microcrystals, by directly observing the early events of enzyme and substrate complex [ E · S ] formation at a single millisecond temporal resolution. The fully saturated enzymatic active site shows the formation of CEF (aminothiazole and dioxotriazine) with the BlaC catalytic residue Ser70 after approximately 50 ms and partial formation of the reaction product, as indicated by the weakened electron density of the CEF density map. ,,, By combining very short and longer delay times and probing intermediates with sufficiently short intervals, it becomes possible to delineate the complete enzymatic reaction steps, from the formation of the enzyme–substrate complex ( E · S ) to the decomposition of E · S , resulting in the regeneration of the free enzyme ( E ) and the release of the reaction product ( P ) (Figure ).…”

“…In addition, we discussed the opportunities and challenges in obtaining crystallographic kinetic information via this method. Mix-and-inject tr -SFX is the only available versatile method applicable to all light nonresponsive proteins and can directly trigger the biochemical reaction of target proteins in crystallo by mixing with its native substrate(s) and probing the formation of reaction intermediates at well-defined and controllable delay times upon substrate binding. , One of the major challenges in MISC tr -SFX, however, is the inevitable requirement for a large number of indexed images of highly isomorphous microcrystals to ensure high accuracy for the reflection intensities within and between the different delay times. ,,− Achieving such a tedious goal may require initially growing homogeneous and highly isomorphous microcrystals and obviously several days of continuous diffraction data collection with reasonable stability in the XFEL sources given the limitation of beamtime availability in XFEL sources. The SACLA, SwissFEL, PAL-XFEL, and LCLS XFEL sources operate at repetition rates in the 10–120 Hz range, which limits the number of delay times (i.e., reaction intermediates) needed to be collected in a given beamtime, which is usually 2 days to full week of data collection. − The European XFEL is the only running facility that operates at high repetition rates up to 4.5 MHz .…”

“…The information required to determine the conformation at a specific delay time (Δ t ) includes the fractional concentration (occupancy) at that specific delay time and the reference structure, which is the protein structure without substrate in the resting state. In other words, the required information is the isomorphous difference electron density (DED) map obtained from the refined structures of the isomorphous pair (reference structure factor amplitudes | F free Obs | for the enzyme-free state and substrate-bound models | F t Obs |, which accounts for the relative accumulation of an intermediate at a specific delay time, Δ t , after reaction initiation) (see refs , for recent accounts on tr -SFX data processing and extraction of transiently formed intermediate states).…”

Changes in an enzyme ensemble during catalysis observed by high-resolution XFEL crystallography

“…However, interpretation of the difference electron density maps produced by time-resolved crystallography experiments can be challenging. Minor conformations populated stochastically rather than in concert during catalysis are especially difficult to identify and model, although recent advances in calculating weighted electron density maps help resolve these conformations ( 35 , 36 ). Because these minor conformations can play important roles in catalysis ( 37 ) and in contributing to protein entropy ( 38 ), it is imperative to develop strategies to enrich them to study important regions of the reaction coordinate.…”